Dataset Information

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

ABSTRACT: AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ?45° and ?35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ?2 on chain B and ?3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.

SUBMITTER: Feng J

PROVIDER: S-EPMC5862418 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

Feng Jinhui J Kweon Ohgew O Xu Haiyan H Cerniglia Carl E CE Chen Huizhong H

Archives of biochemistry and biophysics 20120224 2

AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH in ...[more]

PMID: 22387379

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Project description:The emergence of antimicrobial-resistant and virulent enterococci is a major public health concern. While enterococci are commonly found in food of animal origin, the knowledge on their zoonotic potential is limited. The aim of this study was to determine and compare the antimicrobial susceptibility and virulence traits of Enterococcus faecalis and Enterococcus faecium isolates from human clinical specimens and retail red meat in Slovenia. A total of 242 isolates were investigated: 101 from humans (71 E. faecalis, 30 E. faecium) and 141 from fresh beef and pork (120 E. faecalis, 21 E. faecium). The susceptibility to 12 antimicrobials was tested using a broth microdilution method, and the presence of seven common virulence genes was investigated using PCR. In both species, the distribution of several resistance phenotypes and virulence genes was disparate for isolates of different origin. All isolates were susceptible to daptomycin, linezolid, teicoplanin, and vancomycin. In both species, the susceptibility to antimicrobials was strongly associated with a food origin and the multidrug resistance, observed in 29.6% of E. faecalis and 73.3% E. faecium clinical isolates, with a clinical origin (Fisher's exact test). Among meat isolates, in total 66.0% of E. faecalis and E. faecium isolates were susceptible to all antimicrobials tested and 32.6% were resistant to either one or two antimicrobials. In E. faecalis, several virulence genes were significantly associated with a clinical origin; the most common (31.0%) gene pattern included all the tested genes except hyl. In meat isolates, the virulence genes were detected in E. faecalis only and the most common pattern included ace, efaA, and gelE (32.5%), of which gelE showed a statistically significant association with a clinical origin. These results emphasize the importance of E. faecalis in red meat as a reservoir of virulence genes involved in its persistence and human infections with reported severe outcomes.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

Publications

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

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